United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA 712–C–96–291

June 1996

Health Effects Test

Guidelines

OPPTS 870.1350

Acute Inhalation Toxicity

With Histopathology

‘‘Public Draft’’
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INTRODUCTION

This guideline is one of a series of test guidelines that have been

developed by the Office of Prevention, Pesticides and Toxic Substances,

United States Environmental Protection Agency for use in the testing of

pesticides and toxic substances, and the development of test data that must

be submitted to the Agency for review under Federal regulations.

The Office of Prevention, Pesticides and Toxic Substances (OPPTS)

has developed this guideline through a process of harmonization that

blended the testing guidance and requirements that existed in the Office

of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,

Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the

Office of Pesticide Programs (OPP) which appeared in publications of the

National Technical Information Service (NTIS) and the guidelines published by the Organization for Economic Cooperation and Development

(OECD).

The purpose of harmonizing these guidelines into a single set of

OPPTS guidelines is to minimize variations among the testing procedures

that must be performed to meet the data requirements of the U. S. Environmental Protection Agency under the Toxic Substances Control Act (15

U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act

(7 U.S.C. 136, et seq.).

Public Draft Access Information: This draft guideline is part of a

series of related harmonized guidelines that need to be considered as a

unit. For copies: These guidelines are available electronically from the

EPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines’’ or in paper by contacting the OPP

Public Docket at (703) 305–5805 or by e-mail:

guidelines@epamail.epa.gov.

To Submit Comments: Interested persons are invited to submit comments. By mail: Public Docket and Freedom of Information Section, Office

of Pesticide Programs, Field Operations Division (7506C), Environmental

Protection Agency, 401 M St. SW., Washington, DC 20460. In person:

bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Arlington, VA. Comments may also be submitted electronically by sending

electronic mail (e-mail) to: guidelines@epamail.epa.gov.

Final Guideline Release: This guideline is available from the U.S.

Government Printing Office, Washington, DC 20402 on The Federal Bulletin Board. By modem dial 202–512–1387, telnet and ftp:

fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202–512–0132 for disks

or paper copies. This guideline is also available electronically in ASCII

and PDF (portable document format) from the EPA Public Access Gopher

(gopher.epa.gov) under the heading ‘‘Environmental Test Methods and

Guidelines.’’
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OPPTS 870.1350 Acute inhalation toxicity with histopathology.

(a) Scope—(1) Applicability. This guideline is intended to meet testing requirements of both the Federal Insecticide, Fungicide, and

Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances

Control Act (TSCA) (15 U.S.C. 2601).

(2) Background. This is a new guideline developed in the Office

of Research and Development.

(b) Purpose. In the assessment and evaluation of the potential human

health effects of chemical substances, it is appropriate to test for acute

inhalation toxic effects. The goals of this test are to characterize the exposure-response relationship for sensitive endpoints following acute exposure

and to characterize toxicologic response following acute high exposures.

The latter is of particular concern in relation to spills and other accidental

releases. This testing is designed to determine the gross pathology resulting

from acute inhalation exposure to a substance. In addition, because toxic

effects on the respiratory tract are of particular concern following inhalation exposure, several indicators of respiratory toxicity consisting of

histopathology on fixed tissue and evaluation of cellular and biochemical

parameters in bronchoalveolar lavage fluid should be employed. This acute

testing should be augmented by the respiratory sensory irritation assay in

mice (ASTM, 1984). The respiratory histopathology consists of specialized

techniques to preserve tissues of the respiratory tract in order to allow

detailed microscopic examination to identify adverse effects of chemical

substances on this organ system. The bronchoalveolar lavage is designed

to be a rapid screening test to provide an early indicator of pulmonary

toxicity by examining biochemical and cytologic endpoints of material

from the lungs of animals exposed to potentially toxic chemical substances. The mouse respiratory sensory irritation assay is a non-invasive

assay designed to detect sensory irritation during exposure to a chemical

substance (ASTM, 1984). These acute tests are designed to assess the relationship, if any, between the animals exposed to the test substance, the

incidence and severity of observed abnormalities, gross lesions, body

weight changes, effects on mortality, and any other toxic effects. These

acute tests are not intended to provide a complete evaluation of the

toxicologic effects of a substance, and additional functional and morphological evaluations may be necessary to assess completely the potential

effects produced by a chemical substance. Additional tests may include

longer-term exposures, or more in-depth evaluation of specific organ systems as indicated by signs of toxicity following acute exposure.

(c) Definitions. The following definitions apply to this test guideline.

Aerodynamic diameter refers to the size of particles or aerosols. It

is the diameter of a sphere of unit density that behaves aerodynamically

(has the same settling velocity in air) as the particle of the test substance.

It is used to compare particles of different size, shape and density, and
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to predict where in the respiratory tract such particles may be primarily

deposited.

Exposure response is the relationship between the exposure concentration and the measured response expressed as a group mean in the

case of a continuous variable or as incidence in the case of a quantil variable.

Geometric standard deviation (GSD) is a dimensionless number equal

to the ratio between the mass median aerodynamic diameter (MMAD) and

either 84% or 16% of the diameter size distribution (e.g., MMAD = 2

μm; 84% = 4 μm; GSD = 4/2 = 2.0.) The MMAD, together with the

GSD, describe the particle size distribution of an aerosol.

Lower respiratory tract consists of those structures of the respiratory

tract below the larynx.

The mass geometric mean aerodynamic diameter or the mass median

aerodynamic diameter (MMAD) is the calculated aerodynamic diameter

that divides the particles of an aerosol (a gaseous suspension of fine liquid

or solid particles) in half, based on the weight of the particles. By weight,

50% of the particles will be larger than the MMAD and 50% of the particles will be smaller than the MMAD.

Particle regional deposition is the region of the respiratory tract

where particles of different sizes deposit. Particles between 5 and 30 μm

mainly deposit in the nasopharyngeal region by inertial impaction, particles

between 1 and 5 μm deposit by sedimentation in the tracheal/bronchial/

bronchiolar/alveolar regions, and particles of 1 μm or less deposit in the

alveolar region by diffusion.

Respiratory effects are any adverse effects on the structure or functions of the respiratory system related to exposure to a chemical substance.

Sensory airway irritation is any sign that a chemical substance is

stimulating the nerves of the respiratory tract as identified by the characteristic pause during expiration in the respiratory sensory irritation test.

Toxic effects are any adverse changes (a change that is statistically

or biologically significant) in the structure or function of an experimental

animal as a result of exposure to a chemical substance.

Upper respiratory tract consists of those structures of the respiratory

tract above the larynx.

(d) Principle of the test method. The test substance is administered

to several groups of experimental animals; one concentration level and

duration being used per group. Bronchoalveolar lavage shall be used to

evaluate early effects on the respiratory system by examining changes in

the content of the lavage fluid of the lung. At 24 hours following exposure,
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the animals shall be sacrificed and necropsied, and tissue samples from

the respiratory tract and other major organs will be prepared for microscopic examination. The exposure levels at which significant toxic effects

on the respiratory organ system are produced are compared to those levels

that produce other toxic effects. As triggered by the results of the 4-hour

test, additional exposure periods of 1 hour and 8 hours will be required

to determine the effect of exposure time on the toxicity observed.

(e) Test procedures—(1) Animal selection—(i) Species. In general,

the laboratory rat and mouse should be used. Under some circumstances,

other species, such as the hamster or guinea pig, may be more appropriate,

and if these or other species are used, justification should be provided.

(ii) Strain. If rats and mice are used, the use of the F344 rat and

the B6C3F1 mouse is preferred to facilitate comparison with existing data.

(iii) Age. Young adults shall be used. The weight variation of animals

used in a test should not exceed ± 20 % of the mean weight for each

species.

(iv) Sex. Equal numbers of animals of each sex shall be used for

each dose level. The females shall be nulliparous and nonpregnant.

(v) Health status. Body weight and feed consumption are not sufficient indicators of the health status of animals prior to initiating an inhalation toxicity study. Prior to initiating the study, animals shall be monitored

for known pathogenic rodent viruses as determined by conventional

microbiological assays (e.g., serology). In addition, a few animals shall

be sacrificed prior to exposure, lung lavage fluid shall be evaluated for

polymorphonuclear leukocytes (PMNs), and biochemical parameters shall

be evaluated as in the lung lavage test described in paragraph (e)(12) of

this section to assure the health of the animals. PMNs should be less than

1% of lavage cells. The animals shall be free from pathogens at the start

of exposure.

(2) Number of animals. At least five males and five females shall

be used in each concentration/duration and control group. Animals shall

be randomly assigned to treatment and control groups.

(3) Control groups. A concurrent control group is required for all

tests. The control group shall be a sham treated group. Except for treatment

with the test substance, animals in the control group shall be handled in

a manner identical to the test group animals. Where a vehicle is used to

help generate an appropriate concentration of the substance in the atmosphere, a vehicle control group shall be used.

(4) Concentration level and concentration selection. For the 4-hour

study, at least three concentrations shall be used in addition to the control

group. Ideally, the data generated from the test should be sufficient to
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produce an exposure-effect curve. The concentrations can either be linearly

or logarithmically spaced depending on the anticipated steepness of the

concentration-response curve. A rationale for concentration selection

should be provided to indicate that the selected concentrations will maximally support detection of concentration-effect relationship. The high concentration should be clearly toxic or a limit dose, but should not result

in an incidence of fatalities that would preclude a meaningful evaluation

of the data. The lowest concentration should define a no-observed-adverseeffects level (NOAEL).

(i) Limit dose. The high concentrations need not be greater than 5

mg/L, or for aerosol and particulate concentrations, concentrations that

cannot maintain a size distribution less than or equal to 4 μm (MAD)

(i.e., a particle size distribution that permits deposition throughout the respiratory tract). If a test at an exposure of 5 mg/L (actual concentration

of respirable substance) for 4 hours or, where this is not feasible, the maximum attainable concentration, using the procedures described for this

study, produces no observable toxic effects, then a full study using three

dose levels will not be necessary.

(ii) 1-Hour study. If triggered, three concentrations shall be tested.

These concentrations should allow for the determination of an effect level

and a no-observable-adverse-effect-level.

(iii) 8-Hour study. If triggered, three concentrations shall be tested.

These concentrations should allow for the determination of an effect level

and a no-observable-adverse-effect-level.

(5) Inhalation exposure. Animals can be exposed to the substance

by either a nose only procedure or in a whole body exposure chamber.

(i) Inhalation chambers. The animals shall be tested in inhalation

equipment designed to sustain a dynamic air flow of 12 to 15 air changes

per hour and ensure an adequate oxygen content of at least 19% and an

evenly distributed exposure atmosphere. Where a whole body chamber is

used, its design shall minimize crowding by providing individual caging.

As a general rule, to ensure stability of a chamber atmosphere, the total

‘‘volume’’ of the test animals should not exceed 5% of the volume of

the test chamber.

(ii) Environmental conditions. The temperature at which the test is

performed shall be maintained at 22 °C ( ±2 °C). Ideally, the relative humidity should be maintained between 40% and 60%, but in certain instances (e.g., tests using water as a vehicle), this may not be practical.

(iii) Exposure periodicity. For acute testing, the animals shall be exposed to the test substance for at least 4 hours after the chamber has

reached equilibrium. If triggered by the results of the 4 hour exposure,

additional testing shall be conducted using 1 and 8 hour exposure periods.
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(6) Physical measurements. Measurements or monitoring shall be

made of the following:

(i) The rate of air flow shall be monitored continuously, but shall

be recorded at least every 30 minutes.

(ii) The actual concentrations of the test substance shall be measured

in the breathing zone. During the exposure period, the actual concentrations of the test substance shall be held as constant as practicable, monitored continuously, and recorded at least at the beginning, at an intermediate time, and at the end of the exposure period.

(iii) During the development of the generating system, where appropriate, particle size analysis shall be performed to establish the stability

of aerosol concentrations. During exposure, analysis should be conducted

as often as necessary to determine the consistency of particle size distribution.

(iv) Chemical purity, where appropriate, shall be verified after the

compound is aerosolized, preferably by sampling in the animals’ breathing

zone prior to initiating the study. This is to ensure that purity has not

changed from the stock material to the breathing zone, and that degradation products have not formed.

(v) If the compound is present in a mixture, the mass and composition

of the entire mixture, as well as the principal compound, shall be measured.

(vi) Temperature and humidity shall be monitored continuously, but

shall be recorded at least every 30 minutes.

(7) Food and water during exposure period. Food shall be withheld

during exposure. Water may also be withheld in certain cases.

(8) Observation period. The bronchoalveolar lavage and respiratory

pathology shall be conducted 24 hours following exposure to allow expression of signs of toxicity. There is concern that some latency time will

be required to allow migration of cells and macromolecules into the lungs

following exposure, and that some pathology may require macromolecular

synthesis or degradation before cell damage develops.

(9) Gross pathology. (i) All animals shall be subjected to a full necropsy which includes examination of orifices and the cranial, thoracic,

and abdominal cavities and their contents.

(ii) At least the lungs, liver, kidneys, adrenals, brain, and gonads shall

be weighed wet, as soon as possible after dissection to avoid drying.

(iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future
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histopathological examination: All gross lesions; brain-including sections

of medulla/pons; cerebellar cortex and cerebral cortex; pituitary; thyroid/

parathyroid; thymus; heart; sternum with bone marrow; salivary glands;

liver; spleen; kidneys; adrenals; pancreas; gonads; accessory genital organs

(epididymis, prostrate, and, if present, seminal vesicles); aorta; skin; gall

bladder (if present); esophagus; stomach; duodenum; jejunum; ileum;

cecum; colon; rectum; urinary bladder; representative lymph nodes; thigh

musculature; peripheral nerve; spinal cord at three levels cervical,

midthoracic, and lumbar; and eyes. Tissue preservation for the respiratory

tract is described in paragraph (e)(11) of this section.

(10) Histopathology. The following histopathology shall be performed:

(i) Full histopathology shall be performed on the respiratory tract,

liver and kidney of all animals in the control and high dose groups. The

histopathology of the respiratory tract is described in paragraph (e)(11)

of this section.

(ii) All gross lesions in all animals.

(iii) Target organs in all animals, as indicated by the observations

in the high dose group in this study.

(iv) Archived organs (identified as targets of toxicity from results of

the 90-day study, if a 90-day study is required for this substance) in high

dose animals of the 4-hour acute study should be evaluated to determine

if they are also targets of acute toxicity.

(11) Respiratory tract histopathology. (i) Representative sections of

the respiratory tract shall be examined histologically. These shall include

the trachea, major conducting airways, alveolar region, terminal and respiratory bronchioles, alveolar ducts and sacs, and interstitial tissues.

(ii) Care shall be taken that the method used to kill the animal does

not result in damage to the tissues of the upper or lower respiratory tract.

(iii) Four sections of the nasopharyngeal tissue shall be examined for

histopathologic lesions. This shall include sections through the nasal cavity, and examination of the squamous, transitional, respiratory, and olfactory epithelia. The types and locations of lesions shall be reported using

diagrams of the nose.

(iv) The laryngeal mucosa shall be examined for histopathologic

changes. Sections of the larynx to be examined include the epithelium covering the base of the epiglottis, the ventral pouch, and the medial surfaces

of the vocal processes of the arytenoid cartilages.
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(12) Bronchoalveolar lavage. (i) Animals can be exposed to the substance by either a nose only procedure or in a whole body exposure chamber.

(ii) Care should be taken that the method used to kill the animal results in minimum changes in the fluid of the lungs of the test animals.

(iii) At the appropriate time, the test animals shall be killed and the

heart-lung including trachea removed in bloc. Alternatively, lungs can be

lavaged in situ. If the study will not be compromised, one lobe of the

lungs may be used for lung lavage while the other is fixed for histologic

evaluation. The lungs should be lavaged using physiological saline. The

lavages shall consist of two washes, each of which consists of approximately 80% (e.g., 5 ml in rats and 1 ml in mice) of the total lung volume.

Additional washes merely tend to reduce the concentrations of the material

collected. The lung lavage fluid shall be stored on ice at 5 °C until assayed.

(iv) The following parameters shall be determined in the lavage fluid

as indicators of cellular damage in the lungs: Total protein, cell count,

and percent leukocytes. In addition, a phagocytosis assay using the procedure of Burleson (Burleson et al., 1987; Gilmour and Selgrade, 1993),

paragraphs (h)(1) and (h)(2) of this section, shall be performed to determine macrophage activity.

(13) Combined protocol. The tests described may be combined with

any other toxicity study, as long as none of the requirements of either

are violated by the combination.

(f) Triggered testing. If no adverse effects are seen in the 4-hour

study as compared with controls, no further testing is necessary. If the

4-hour study shows positive effects in histopathology of the

bronchoalveolar lavage, a 1-hour study and an 8-hour study shall be conducted. Only those systems showing positive results in the 4-hour study

must be pursued in the follow-up 1-hour and 8-hour studies.

(g) Data reporting and evaluation. The final test report must include

the following information:

(1) Description of equipment and test methods. A description of

the general design of the experiment and any equipment used shall be

provided. This shall include a short justification explaining any deviations

from protocol.

(i) Description of exposure apparatus, including design, type, dimensions, source of air, system for generating particulates, aerosols, gasses,

and vapors, method of conditioning air, treatment of exhaust air, and the

method of housing animals in a test chamber.

(ii) Description of the equipment for measuring temperature, humidity, and particulate aerosol concentration and size.
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(iii) Exposure data shall be tabulated and presented with mean values

and measure of variability (e.g., standard deviation) and should include:

(A) Airflow rates through the inhalation equipment.

(B) Temperature and humidity of air.

(C) Nominal concentration (total amount of test substance fed into

the inhalation equipment divided by the volume of air).

(D) Actual concentration in test breathing zone.

(E) Particle size distribution (e.g., MMAD with GSD), where appropriate.

(2) Results—(i) General group animal data. The following information must be arranged by test group exposure level.

(A) Number of animals exposed.

(B) Number of animals dying.

(C) Number of animals showing overt signs of toxicity.

(D) Pre- and post-exposure body weight change in animals, and

weight change during the observation period.

(ii) Counts and incidence of gross alterations observed at necropsy

in the test and control groups. Data shall be tabulated to show:

(A) The number of animals used in each group and the number of

animals in which any gross lesions were found.

(B) The number of animals affected by each different type of lesion,

and the locations and frequency of each type of lesion.

(iii) Counts and incidence of general histologic alterations in the

test group. Data shall be tabulated to show:

(A) The number of animals used in each group and the number of

animals in which any histopathologic lesions were found.

(B) The number of animals affected by each different type of lesion,

and the locations, frequency, and average grade of each type of lesion.

(iv) Counts and incidence of respiratory histopathologic alterations by the test group. The nasal lesions report shall be accompanied

by diagrams of the locations of the lesions. Data shall be tabulated to

show:

(A) The number of animals used in each group and the number of

animals in which any lesion was found.
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(B) The number of animals affected by each different type of lesion,

and the locations, frequency, and average grade of each type of lesion.

(v) Results of the bronchoalveolar lavage study. Data shall be tabulated to show:

(A) The amount of administered lavage fluid and recovered lavage

fluid for each test animal.

(B) The magnitude of change of biochemical and cytologic indices

in lavage fluids at each test concentration for each animal.

(C) Results shall be quantified as amount of constituent/mL of lavage

fluid. This assumes that the amount of lavage fluid recovered is a representative sample of the total lavage fluid.

(3) Evaluation of data. The findings from this acute study should

be evaluated in the context of preceding and/or concurrent toxicity studies

and any correlated functional findings. The evaluation shall include the

relationship between the concentrations of the test substance and the presence or absence, incidence, and severity of any effects. The evaluation

should include appropriate statistical analyses, for example, parametric

tests for continuous data and non-parametric tests for the remainder.

Choice of analyses should consider tests appropriate to the experimental

design, including repeated measures. The report must include concentration-effect curves for the bronchoalveolar lavage and tables reporting observations at each concentration level for necropsy findings and gross, general, and respiratory system histopathology.

(h) References. The following references should be consulted for additional background material on this test guideline.

(1) Burleson, G.R., Fuller, L.B., Me’nache, M.G., and Graham, J.A.

‘‘Poly (I): poly (C)-enhanced alveolar peritoneal macrophage phagocytosis: Quantification by a new method utilizing fluorescent beads.’’ Proceedings of the Society of Experimental Biology and Medicine. 184:468–476

(1987).

(2) Gilmour, G.I., and Selgrade, M.K. ‘‘A comparison of the pulmonary defenses against streptococcal infection in rats and mice following

O3 exposure: Differences in disease susceptibility and neutrophil recruitment.’’ Toxicology and Applied Pharmacology. 123:211–218 (1993).

(3) Henderson, R.F., Benson, J.M., Hahn, F.F., Hobbs, C.H., Jones,

R.K., Mauderly, J.L., McClellan, R.O., and Pickrell, J.A. ‘‘New approaches for the evaluation of pulmonary toxicity: Bronchoalveolar lavage

fluid analysis.’’ Fundamental and Applied Toxicology. 5:451–458 (1985).

(4) Henderson, R.F. ‘‘Use of bronchoalveolar lavage to detect lung

damage.’’ Environmental Health Perspectives. 56:115–129 (1984).
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(5) Henderson, R.F., Rebar, A.H., Pickrell, J.A., and Newton, G.J.

‘‘Early damage indicators in the lung. III. Biochemical and cytological

response of the lung to inhaled metal salts.’’ Toxicology and Applied Pharmacology. 50:123–136 (1979).

(6) Renne, R.A., Gideon, K.M., Miller, R.A., Mellick, P.W., and

Grumbein, S.L. ‘‘Histologic methods and interspecies variations in the laryngeal histology of F344/N rats and B6C3F1 mice.’’ Toxicology and Pathology. 20:44–51 (1992).

(7) Young, J.T. ‘‘Histopathologic examination of the rat nasal cavity.’’ Fundamental and Applied Toxicology. 1:309–312 (1981).